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Optimisation for a viable [3H]PAF binding to human platelet membranes
PROSTAGLANDINS
OPTIMISATION FOR A VIABLE 13H1PAF BINDING TO HUNAN PLATELET MEMBRANES. Tahraoui I_, Floch A., Cavero I., CRV, RhBne-Poulenc Sante, BP 14, 94403 Vitry-sur-Seine. France. PAF has been suggested to intervene in numerous pathological processes such as inflammation and allergy. This study was aimed to the optimisation of 13~1~~~ binding to human platelet membranes. Blood (withdrawn from the cubital vein of 2.5-50 yrs old healthy volunteers, who abstained taking any medication during the previous 10 days) was centrifuged for the separation of platelets (PRPl which were subsequently lysed and homogenized. The resulting suspension was centrifuged and the obtained membranes were finally homogenized in an assay buffer (50 mM Tris HCl, 1 mM EDTA, 5 mM MgClz, pH=?.4 and 0.25% BSA). r3H1PAF (0.017 + 1.5 nM1 plus 1 NM of cold PAF-0s or 59227 RP, a PAF antagonist, was added to aliquots of this membrane preparation. After a 60 min incubation period at 25"C, the reaction was stopped by a rapid filtration procedure and the bound [sH]PAF counted by liquid scintillation spectroscopy. The binding of L3HlPAF was found to be saturable, specific and entirely displaceable by cold PAF or 59227 RP. Scatchard analysis revealed a single class of binding sites with a Kd of 0.075+0.005 nM and Bmax of 202f14 fmol/mg protein (n=241. A variety of classical pharmacological agents were unable to interfere with this binding. However, ;he PAF antagonists CV 6209, 59227 RP, WEB 2086, kadsurenone displaced fully [ HIPAF with Ki values of 1.8iO.3, 1.9f0.7, 39.626.2 and 231f41 nM (n=41, respectively. A good correlation (r = 0.851 was demonstrated between the inhibition of f3HIPAF binding and PAF-induced platelet aggregation for a series of 23 PAF antagonists of diverse chemical structures. Thus, the characterised binding procedure may be envisaged for studying possible changes in r3H1PAF binding parameters in patients suffering from disease states that may be causally related to an abnormal PAF liberation.
NEUTROPHILS INDUCE PLATELET ACTIVATION IN RABBIT AND HUMAN : A ROLE FOR PAF-ACETHER ONLY IN RABBIT.-, Chignard M., Joseph, D. and Vargaftig B.B., Uniteassociee IP/INSERM U285, lnstitut Pasteur, 25 rue du Dr. Roux, 75015, Paris, France. Purified rabbit neutrophils and platelets were co-incubated at the physiolological concentrations of 5 x 10’ and 5 x IO* per ml respectively. Addition of the synthetic chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) concentration-dependently induced platelet aggregation and secretion with a maximal and significant effect at 0.1 PM. Aggregation started after a time lag of about 60 s and reached its maximal at 3 min. Washed human platelets (2 x log/ml) in presence of autologous purified neutrophils (5 x IO6 /ml) did also aggregate and release serotonin when challenged with FMLP. As observed with rabbit cells, the range of active FMLP concentrations was similar to that used for activating neutrophils alone (as measured by 6glucuronidase release) and for both species, addition of FMLP to platelets alone failed to trigger activation. This shows that activation of platelets by FMLP is a process mediated by neutrophils. Nonetheless, it appears that the mechanisms of neutrophil-induced platelet activation are different in each species. Firstly, the PAF-acether antagonist, BN 52021 (100 PM), supressed by 70-80 % the cooperation between rabbit cells but was unable to affect significantly the cooperation between human cells. Secondly, supernatants of FMLP-stimulated rabbit neutrophils induced platelet activation only when bovine serum albumin, a PAF-acether carrier, was present which was not the case for supernatants of FMLP-stimulated human neutrophils. Thirdly, supernatants of human neutrophils activated without preincubation with cytochalasin B, a chemical not required for PAF-acether synthesis, were inactive whereas supernatants from rabbit neutrophils, processed under the same conditions, were still effective. Our results clearly show that the cooperation of human cells does not involve PAF-acether participation whereas the cooperation between rabbit cells is largely due to PAF-acether. (Part of this work was done in collaboration with M.A. Selak and J.B. Smith, Temple U., Philadelphia).
MAY 1988 VOL. 35 NO. 5
OPTIMISATION FOR A VIABLE 13H1PAF BINDING TO HUNAN PLATELET MEMBRANES. Tahraoui I_, Floch A., Cavero I., CRV, RhBne-Poulenc Sante, BP 14, 94403 Vitry-sur-Seine. France. PAF has been suggested to intervene in numerous pathological processes such as inflammation and allergy. This study was aimed to the optimisation of 13~1~~~ binding to human platelet membranes. Blood (withdrawn from the cubital vein of 2.5-50 yrs old healthy volunteers, who abstained taking any medication during the previous 10 days) was centrifuged for the separation of platelets (PRPl which were subsequently lysed and homogenized. The resulting suspension was centrifuged and the obtained membranes were finally homogenized in an assay buffer (50 mM Tris HCl, 1 mM EDTA, 5 mM MgClz, pH=?.4 and 0.25% BSA). r3H1PAF (0.017 + 1.5 nM1 plus 1 NM of cold PAF-0s or 59227 RP, a PAF antagonist, was added to aliquots of this membrane preparation. After a 60 min incubation period at 25"C, the reaction was stopped by a rapid filtration procedure and the bound [sH]PAF counted by liquid scintillation spectroscopy. The binding of L3HlPAF was found to be saturable, specific and entirely displaceable by cold PAF or 59227 RP. Scatchard analysis revealed a single class of binding sites with a Kd of 0.075+0.005 nM and Bmax of 202f14 fmol/mg protein (n=241. A variety of classical pharmacological agents were unable to interfere with this binding. However, ;he PAF antagonists CV 6209, 59227 RP, WEB 2086, kadsurenone displaced fully [ HIPAF with Ki values of 1.8iO.3, 1.9f0.7, 39.626.2 and 231f41 nM (n=41, respectively. A good correlation (r = 0.851 was demonstrated between the inhibition of f3HIPAF binding and PAF-induced platelet aggregation for a series of 23 PAF antagonists of diverse chemical structures. Thus, the characterised binding procedure may be envisaged for studying possible changes in r3H1PAF binding parameters in patients suffering from disease states that may be causally related to an abnormal PAF liberation.
NEUTROPHILS INDUCE PLATELET ACTIVATION IN RABBIT AND HUMAN : A ROLE FOR PAF-ACETHER ONLY IN RABBIT.-, Chignard M., Joseph, D. and Vargaftig B.B., Uniteassociee IP/INSERM U285, lnstitut Pasteur, 25 rue du Dr. Roux, 75015, Paris, France. Purified rabbit neutrophils and platelets were co-incubated at the physiolological concentrations of 5 x 10’ and 5 x IO* per ml respectively. Addition of the synthetic chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) concentration-dependently induced platelet aggregation and secretion with a maximal and significant effect at 0.1 PM. Aggregation started after a time lag of about 60 s and reached its maximal at 3 min. Washed human platelets (2 x log/ml) in presence of autologous purified neutrophils (5 x IO6 /ml) did also aggregate and release serotonin when challenged with FMLP. As observed with rabbit cells, the range of active FMLP concentrations was similar to that used for activating neutrophils alone (as measured by 6glucuronidase release) and for both species, addition of FMLP to platelets alone failed to trigger activation. This shows that activation of platelets by FMLP is a process mediated by neutrophils. Nonetheless, it appears that the mechanisms of neutrophil-induced platelet activation are different in each species. Firstly, the PAF-acether antagonist, BN 52021 (100 PM), supressed by 70-80 % the cooperation between rabbit cells but was unable to affect significantly the cooperation between human cells. Secondly, supernatants of FMLP-stimulated rabbit neutrophils induced platelet activation only when bovine serum albumin, a PAF-acether carrier, was present which was not the case for supernatants of FMLP-stimulated human neutrophils. Thirdly, supernatants of human neutrophils activated without preincubation with cytochalasin B, a chemical not required for PAF-acether synthesis, were inactive whereas supernatants from rabbit neutrophils, processed under the same conditions, were still effective. Our results clearly show that the cooperation of human cells does not involve PAF-acether participation whereas the cooperation between rabbit cells is largely due to PAF-acether. (Part of this work was done in collaboration with M.A. Selak and J.B. Smith, Temple U., Philadelphia).
MAY 1988 VOL. 35 NO. 5